Previous research suggested that Pseudomonas spp. may attack the pyrrolidine ring of nicotine in a waysimilar to mammalian metabolism, resulting in the formation of pseudooxynicotine, the direct precursor of apotent tobacco-specific lung carcinogen. In addition, the subsequent intermediates, 6-hydroxy-3-succinoylpyridine(HSP) and 2,5-dihydroxypyridine (DHP) in the Pseudomonas nicotine degradation pathway are twoimportant precursors for drug syntheses. However, there is little information on the molecular mechanism fornicotine degradation via the pyrrolidine pathway until now. In this study we cloned and sequenced a 4,879-bpgene cluster involved in nicotine degradation. Intermediates N-methylmyosmine, pseudooxynicotine, 3-succinoylpyridine,HSP, and DHP were identified from resting cell reactions of the transformant containing the genecluster and shown to be identical to those of the pyrrolidine pathway reported in wild-type strain Pseudomonasputida S16. The gene for 6-hydroxy-3-succinoylpyridine hydroxylase (HSP hydroxylase) catalyzing HSP directlyto DHP was cloned, sequenced, and expressed in Escherichia coli, and the purified HSP hydroxylase (38 kDa)is NADH dependent. DNA sequence analysis of this 936-bp fragment reveals that the deduced amino acidshows no similarity with any protein of known function.